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Transcriptome-wide mapping of N3-methylcytidine modification at single-base resolution

ABSTRACT: Transcriptome-wide mapping of N3-methylcytidine modification at single-base resolution

PROVIDER: PRJNA1212065 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR32037328_1.fastq.gz	Fastqsanger.gz
	SRR32037328_2.fastq.gz	Fastqsanger.gz
	SRR32037329_1.fastq.gz	Fastqsanger.gz
	SRR32037329_2.fastq.gz	Fastqsanger.gz
	SRR32037330_1.fastq.gz	Fastqsanger.gz

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Transcriptome-wide mapping of N3-methylcytidine modification at single-base resolution

Project description:3-Methylcytidine (m3C), a prevalent modification of tRNAs, was recently identified in eukaryotic mRNAs. However, its precise distribution and formation mechanisms in mRNAs remain elusive. Here we develop a novel approach, m3C immunoprecipitation and sequencing (m3C-IP-seq), utilizing antibody enrichment to profile the m3C methylome at single-nucleotide resolution. m3C-IP-seq captures 12 m3C modification sites in cytoplasmic tRNA isoacceptors and 2 in mitochondrial tRNA isoacceptors. Moreover, m3C-IP-seq permits the comprehensive profiling of m3C sites in mRNAs and lncRNAs, with their presence reliant on a nuclear isoform of METTL8. A significant proportion of m3C sites is concentrated in the 3’ untranslated region (3’ UTR) of mRNAs. Additionally, m3C methylation is dynamic and responds to hypoxia. Functionally, m3C formation in the 3’ UTR is associated with mRNA degradation. Collectively, our data demonstrate the widespread presence of m3C modification in the human transcriptome and provide a resource for functional studies of m3C-mediated RNA metabolism.

2025-03-19 | GSE287299 | GEO

Transcriptome-wide mapping of N3-methylcytidine modification at single-base resolution.

Project description: Not available

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Detection of N3-methylcytidine in RNA at single base resolution using Protoscript II to read through and generate high mutation rates [m3C_tRNA]

Project description:N3-methylcytidine (m3C), an RNA modification involving methylation of cytidine at the Watson-Crick face, has been identified in human tRNA, mitochondrial tRNA (mt-tRNA), and mRNA. In tRNA and mt-tRNA, it has been shown to play important roles in tRNA structure and translation. m3C remains understudied in mRNA and other low-abundance RNAs due to its markedly lower levels, the lack of high-resolution techniques to reproducibly map its location, and under-characterization of its effector proteins on these RNA types. Here we describe a new approach which utilizes the high readthrough and mutation rates of Protoscript II (PSII) at m3C sites in combination with AlkB demethylation to detect m3C at single-base resolution. We first validate the method on synthetic probes. Then, using this method, we successfully identify the known m3C sites on tRNA and mt-tRNA and show that none of the m3C sites identified in cytosolic tRNA are likely METTL8 substrates. In contrast, the two m3C sites detected in mt-tRNA are installed by METTL8 since they are sensitive to METTL8 knockdown (KD) and over expression (OE). These results demonstrate that the method can be used to investigate changes in the m3C level in many sites using an unbiased approach. Overall, this study introduces a convenient high-resolution method for m3C mapping with high readthrough rates, paving the way for future mechanistic studies on the biological function of m3C and its effector proteins.

2026-07-17 | GSE329484 | GEO

Detection of N3-methylcytidine in RNA at single base resolution using Protoscript II to read through and generate high mutation rates [m3C_screen]

2026-07-17 | GSE329485 | GEO

Detection of N3-methylcytidine in RNA at single base resolution using Protoscript II to read through and generate high mutation rates [m3C_screen]

Project description:Detection of N3-methylcytidine in RNA at single base resolution using Protoscript II to read through and generate high mutation rates [m3C_screen]

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Detection of N3-methylcytidine in RNA at single base resolution using Protoscript II to read through and generate high mutation rates [m3C_tRNA]

Project description:Detection of N3-methylcytidine in RNA at single base resolution using Protoscript II to read through and generate high mutation rates [m3C_tRNA]

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N3-Kethoxal-seq for genome-wide RNA structure mapping in vivo

Project description:We established N3-Kethoxal molecule to probe RNA secondary structure in vivo at transcriptome level.

2020-02-22 | GSE122096 | GEO

Herbaspirillum hiltneri N3 strain:N3

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Genome-wide mapping of epigenetic modification of 5-formylcytosine at single-base resolution by chemical labeling enrichment and deamination sequencing

Project description:DNA cytosine methylation (5-methylcytosine, 5mC) is a predominant epigenetic modification that plays critical roles in a variety of biological and pathological processes in mammals. In active DNA demethylation, the ten-eleven translocation (TET) dioxygenases can sequentially oxidize 5mC to generate three modified forms of cytosine, 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Beyond being a demethylation intermediate, recent studies have shown that 5fC has regulatory functions in gene expression and chromatin organization. While some methods have been developed to detect 5fC, genome-wide mapping of 5fC at base resolution are still highly desirable. Herein, we propose a Chemical Labeling Enrichment and Deamination sequencing (CLED-seq) method for detecting 5fC in genomic DNA at single-base resolution. The CLED-seq method utilizes selective labeling and enrichment of 5fC-containing DNA fragments, followed by deamination mediated by A3A and sequencing. In the CLED-seq process, while all C, 5mC, and 5hmC are interpreted as T during sequencing, 5fC is still read as C, enabling the precise detection of 5fC in DNA. Using the proposed CLED-seq method, we accomplished genome-wide mapping of 5fC in mouse embryonic stem cells. The mapping study revealed that promoter regions enriched with 5fC overlapped with H3K4me1, H3K4me3, and H3K27ac marks. These findings suggest a correlation between 5fC marks and active gene expression in mouse embryonic stem cells. In conclusion, CLED-seq is a straightforward, bisulfite-free method that offers a valuable tool for detecting 5fC in genomes at a single-base resolution.

2023-08-31 | GSE239943 | GEO

Base-resolution analysis of deoxyuridine at genome scale based on artificial incorporation modified nucleobase

Project description:Deoxyuridine (dU) in DNA can result from deamination of cytosine and dUMP misincorporation. The easy single-nucleotide resolution assay for dU are crucial to understanding its role in genome. Herein, we present a new concept of “base substitution” for accurate single-nucleotide resolution profiling of dU. The method termed AI-Seq (Artificial Incorporation of a modified nucleobase for sequencing) was developed using artificial cytosine (N3-C) to replace dU. After the artificial base construction, dU can read as cytosine during polymerase chain reaction assay. AI-seq was validated on synthetic DNA and then applied to the genome of HEK293T cell line. Collectively, the “base substitution” provides a novel approach for generating comprehensive information about the distribution of dU and can be potentially adapted to detect other epigenetic modifications.

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